WO2013071678A1 - Kit de dosage immunoenzymatique compétitif d'antigènes par marqueurs polypeptides du cancer du foie - Google Patents

Kit de dosage immunoenzymatique compétitif d'antigènes par marqueurs polypeptides du cancer du foie Download PDF

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Publication number
WO2013071678A1
WO2013071678A1 PCT/CN2012/000221 CN2012000221W WO2013071678A1 WO 2013071678 A1 WO2013071678 A1 WO 2013071678A1 CN 2012000221 W CN2012000221 W CN 2012000221W WO 2013071678 A1 WO2013071678 A1 WO 2013071678A1
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WO
WIPO (PCT)
Prior art keywords
gly
liver cancer
polypeptide
polypeptide marker
monoclonal antibody
Prior art date
Application number
PCT/CN2012/000221
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English (en)
Chinese (zh)
Inventor
魏开华
肖汉族
周晓明
付海媛
原剑
侯利平
杨保安
孙云波
黄亚娟
郑俊杰
甄蓓
张拓
王东茂
Original Assignee
北京正旦国际科技有限责任公司
湖南金健药业有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by 北京正旦国际科技有限责任公司, 湖南金健药业有限责任公司 filed Critical 北京正旦国际科技有限责任公司
Publication of WO2013071678A1 publication Critical patent/WO2013071678A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a biologic polypeptide marker antigen competitive enzyme-linked immunoassay kit and a detection method thereof. Background technique
  • liver cancer surgery With the maturity and development of liver cancer surgery, the treatment methods and means of liver cancer are increasingly abundant, and are no longer limited to a single surgical treatment. Liver resection and liver transplantation are still the main methods for treatment of liver cancer patients, but minimally invasive treatment, percutaneous transcatheter arterial chemoembolization and other technical methods, as an important supplement to surgical treatment, have also been widely used, they are created for patients. Surgical treatment opportunities, reducing or reducing tumor recurrence and metastasis have played a role in extending the survival time of patients. Despite this, patients with liver cancer have insidious onset, no obvious symptoms, and most patients have lost the opportunity to undergo radical surgery. Therefore, "early detection, early diagnosis, early treatment” is an important measure to reduce cancer mortality. Screening and establishing highly sensitive, highly specific serological diagnostic techniques for early diagnosis of cancer, providing early warning screening for cancer, is of great significance and great market prospects.
  • AFP alpha-fetoprotein
  • HCC liver cancer
  • the peptide in serum is different from the protein in serum, and its molecular weight is lower than 1000OOD, and the structure is simple and varied. Whether it is possible to use the current routine clinical means for testing is still being explored.
  • a serum polypeptide is a degradation product of a serum albumin or a gene directly encoded product.
  • the metabolism of the protein changes, resulting in a corresponding change in the polypeptide, thereby establishing a correlation between the polypeptide and the disease. Therefore, the polypeptide can be diagnosed like a protein.
  • some diseases have been diagnosed using peptide markers.
  • the early detection kit for tumor based on serum polypeptide antigen is one of the most important early diagnosis techniques for tumors.
  • single markers in the clinical field always have the disadvantages of low specificity and low positive rate. Especially the detection rate of early tumors is not high. It is imperative to detect multiple markers. However, there is no good means. Achieve simultaneous detection.
  • Josep Villanueva et al. studied 32 cases of prostate cancer, 21 cases of breast cancer and 20 cases of bladder cancer serum peptides, and found that 14, 10 and 58 can be used as tumor marker polypeptides for prostate cancer, breast cancer and bladder cancer, respectively.
  • the prediction accuracy is 100%.
  • the abundant peptides present in serum provide abundant marker resources for tumor early warning and early diagnosis.
  • the object of the present invention is to provide a kit for detecting a liver cancer polypeptide marker antigen competitive enzyme-linked immunoassay and a method for detecting the same.
  • the invention provides a liver cancer polypeptide marker antigen competitive enzyme-linked immunosorbent assay kit, which comprises the monoclonal antibody secreted by the monoclonal antibody hybridoma cell line AP0105 of the anti-human primary liver cancer polypeptide marker with the accession number CGMCC No. 5269.
  • hybridoma cell line is a polypeptide marker antigen (sequence is Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-
  • polypeptide 5 The polypeptide of Gln, designated as polypeptide 5), is conjugated to the carrier protein keyhole limpet hemocyanin (KLH) immunogen, immunized to BALB/C mice, and then screened for hybridoma cell lines.
  • KLH keyhole limpet hemocyanin
  • the monoclonal antibody hybridoma cell line AP0105 for anti-human primary liver cancer polypeptide marker provided by the present invention has been on the ordinary microbiology center of China Microbial Culture Collection Management Committee on September 27, 2011 (Address: Beichen West, Chaoyang District, Beijing) No. 3, No. 1 Road, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) Deposit, deposited as CGMCC No. 5269.
  • the monoclonal antibody is an enzyme-labeled monoclonal antibody, and preferably, the labeled enzyme may be horseradish peroxidase.
  • the liver cancer polypeptide marker antigen competitive enzyme-linked immunosorbent assay kit of the present invention may further comprise a coating solution, 5% skim milk powder, TMB color developing solution, 2M sulfuric acid, and 96-well enzyme-linked plate.
  • the invention provides a method for detecting a liver cancer polypeptide marker antigen, comprising the steps of:
  • the method specifically includes the following steps:
  • the sample to be tested is combined with 0.3 g/mL of HRP-labeled polypeptide antibody at a volume ratio of 1: 1 at 37 °C for 0.5-1 hour; at the same time, PBS is used instead of the sample to be tested as a negative pair.
  • the invention also provides the use of the above enzyme-linked immunoassay kit for detecting liver cancer polypeptide marker antigen.
  • the monoclonal antibody provided by the invention has strong specificity against the polypeptide marker antigen;
  • the method for detecting the peptide marker antigen is simple and rapid, and is convenient for clinical detection; and can be detected by a high-throughput, low-cost enzyme-linked detector.
  • the invention combines polypeptide antibody with ELSIA technology, can simultaneously detect multiple biomarker groups, and can be used for large-scale sample detection, and is an ideal tool for verifying serum markers and for clinical detection.
  • Fig. 1 is a graph showing the ROC curve of liver cancer and normal serum in the present invention. detailed description
  • Example 1 Method for preparing polypeptide marker antigen monoclonal antibody
  • the recombinant polypeptide 5 conjugated to the carrier protein KLH was used as an immunogen to immunize BALB/C mice; its full-length sequence is: Asn-Leu-Gly-His-Gly-His-Lys-Hi-Glu-Arg-Asp -Gln-Gly-His-Gly-His-Gln.
  • polypeptide standard solution ⁇ g/ml coating solution of synthetic peptide 5;
  • TMB color developing solution purchased from Beijing Kangwei Century Biotechnology Co., Ltd.;
  • Samples 60 clinically diagnosed liver cancer serum and normal serum.
  • the monoclonal antibody provided by the invention has strong specificity against the polypeptide marker antigen; the method for detecting the polypeptide marker antigen is simple and rapid, and is convenient for clinical detection; and can be detected by a high-throughput and low-cost enzyme-linked detector. Multiple biomarker groups can be detected at the same time, and it can be used for large-scale sample detection, which reduces costs. It is an ideal tool for verifying serum markers and for clinical detection, and has important economic value and application prospects.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Hospice & Palliative Care (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un kit de dosage immunoenzymatique compétitif d'antigènes par marqueurs polypeptides du cancer du foie, comportant : un anticorps monoclonal sécrété par des lignes de cellules d'hybridomes AP0105 d'anticorps monoclonal d'un marqueur polypeptide artificiel d'antigène du cancer du foie caractérisé par le numéro de conservation CGMCC No.5269 et une substance polypeptide standard en concurrence avec le marqueur polypeptide dans un objet à tester, la séquence du polypeptide étant : Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp-Gln-Gly-His-Gly-His-Gln.
PCT/CN2012/000221 2011-11-14 2012-02-21 Kit de dosage immunoenzymatique compétitif d'antigènes par marqueurs polypeptides du cancer du foie WO2013071678A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110359486.4A CN102520175B (zh) 2011-11-14 2011-11-14 一种肝癌多肽标志物抗原竞争性酶联免疫检测试剂盒
CN201110359486.4 2011-11-14

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Publication Number Publication Date
WO2013071678A1 true WO2013071678A1 (fr) 2013-05-23

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PCT/CN2012/000221 WO2013071678A1 (fr) 2011-11-14 2012-02-21 Kit de dosage immunoenzymatique compétitif d'antigènes par marqueurs polypeptides du cancer du foie

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CN (1) CN102520175B (fr)
WO (1) WO2013071678A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN102998451B (zh) * 2012-11-22 2015-02-04 北京正旦国际科技有限责任公司 与肝病进程相关的检测试剂盒及其应用
CN102998452B (zh) * 2012-11-22 2014-09-10 北京正旦国际科技有限责任公司 结肠癌多肽标志物Pep5酶联免疫检测试剂盒的制备方法

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KR100545064B1 (ko) * 2003-05-26 2006-01-24 김현기 인간 원암단백질에 특이적인 항체를 포함하는 간경변증진단 키트
JP5424331B2 (ja) * 2007-10-18 2014-02-26 国立大学法人 鹿児島大学 肝疾患診断用バイオマーカー
CN101672850A (zh) * 2009-01-05 2010-03-17 中山大学 肝硬化与早期肝癌鉴别诊断试剂盒及血清clusterin在制备该试剂盒中的应用
US20120183981A1 (en) * 2009-08-28 2012-07-19 Inova Diagnostics, Inc. Methoed for detecting circulating cartilage oligomeric matrix protein in the diagnosis and monitoring of cirrhosis
CN201477095U (zh) * 2009-09-02 2010-05-19 同昕生物技术（北京）有限公司 灵敏快速的肝硬化和肝癌磁珠酶免法诊断试剂盒与磁珠
CN102062780A (zh) * 2010-11-23 2011-05-18 北京正旦国际科技有限责任公司 一种多肽免疫试剂盒及其检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VILANUEVA, J. ET AL.: "Differential exoprotease activities confer tumor-specific serum peptidome patterns", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 116, no. 1, 31 January 2006 (2006-01-31), pages 271 - 284, XP002434651 *
YUAN, JIAN ET AL.: "Determination of Serum Peptide Biomarkers in Primary Hepatocellular Carcinoma by Immunocapture Using Peptide Antibodies and Mass Spectrometry", CHINESE JOURNAL OF ANALYTICAL CHEMISTRY, vol. 39, no. 8, 31 August 2011 (2011-08-31), pages 1129 - 1133 *

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CN102520175A (zh) 2012-06-27

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